Small molecule inhibitors of cancer stem cells and mesenchymal cancer types

ABSTRACT

The present disclosure provides compounds, their pharmaceutical compositions, and methods of their use for treating mesenchymally-derived or mesenchymally transformed cancers, such as breast cancers and sarcomas, and for treating diseases or disorders that are characterized by the expression of vimentin.

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/578,897, filed on Oct. 30, 2017, whichapplication is incorporated by reference as if fully set forth herein.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under grant numberCA200970 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Cancer stem cells (CSCs) play critical roles in cancer progression andtreatment resistance. A wealth of literature has demonstrated thatepithelial-mesenchymal transition (EMT) processes are responsible forgenerating the CSC pool in multiple cancer types, including breastcancer, and for endowing cancer cells with metastatic competence.

Current breast cancer standards of care include hormonal treatments(e.g. tamoxifen, anastrozole, letrozole), HER-2 targeted therapies(trastuzumab, lapatinib), and conventional chemotherapeutic agents (e.g.docetaxel, doxorubicin, capecitabine). Yet, none of these therapiestarget the CSC population and, therefore, in addition to theirrespective side effects, the therapies have limited efficacy inpromoting relapse free survival and inhibiting metastasis. In thiscontext, resistance to chemotherapy and metastasis are the major causesfor breast cancer-related mortality.

Soft tissue sarcomas (STS) are a rare and heterogeneous class of tumors,grouped solely by their mesenchymal origin (I. Zambo et al. Cesk Patol50 (2014) 64-70). Current standards of care for STS patients arecurrently limited to surgical resection and treatment with typicalDNA-damaging chemotherapies (e.g., doxorubicin, ifosfamide) orradiation, interventions which yield five-year median survival rates ofonly 50% for late stage STS patients (M. J. Nathenson et al. CancerChemother. Pharmacol. 78 (2016) 895-919). Additionally, only a handfulof targeted therapies have been approved or are under investigation forSTS indications (A. R. Dancsok et al. Oncotarget 8(4) (2017) 7068-7093).

Although a host of transcription factors and mitogens are capable ofinducing EMT in breast cancer, the presence of transition factorForkhead Box C2 (FOXC2) is reported for effective EMT via any stimulusstudied to date (S. A. Mani et al., Proc. Natl. Acad. Sci. USA 104(2007) 10069-10074; B. G. Hollier et al., Cancer Res. 73 (2013)1981-1982). Additionally, exogenous expression of FOXC2 is reported toendow non-metastatic cancer cell lines with metastatic potential(Hollier (2013)).

SUMMARY

The present disclosure provides compounds that are useful as selectivelycytotoxic agents against cells of the EMT-CSC phenotype. In addition,the compounds also inhibit the growth of mesenchymally-transformedcancer cells by binding to and interfering with the organization andphosphorylation of vimentin during mitosis. In contrast to the manynaturally- and synthetically-derived compounds targeting microtubules,the compounds disclosed herein target the intermediate filament proteinvimentin to promote mitotic catastrophe. Because vimentin expression ismanifested in mesenchymal as well as endothelial and haemopoietic celltypes, the compounds moreover constitute genotype-selectivechemotherapeutics for the treatment of mesenchymal cancers and otherdisorders, such as those involving non-mesenchymal cell types, thatinvolve vimentin expression. The disclosure thus provides in oneembodiment a method for treating a patient suffering from amesenchymally-derived or mesenchymally-transformed cancer, comprisingadministering to the subject a therapeutically effective amount of acompound, or a pharmaceutically acceptable salt there, according to anyone of Formulae I-IV:

In Formula I compounds, R^(1A) in each instance is independentlyselected from the group consisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, andC₁-C₆-alkoxy, halo, CN, —S—C₁-C₆-alkyl, and —C(O)N(R^(2A))₂.

R^(2A) in each instance is independently selected from H andC₁-C₆-alkyl;

Each ---, if present, represents a single bond, and n is 0, 1, 2, or 3.

Het^(A) is 6-membered monocyclic or 9- to 10-membered bicyclicheteroaryl, wherein 1 to 3 ring members are N;

Het^(A) and any alkyl, alkenyl, alkoxy, and aryl is optionallysubstituted with 1-3 substituents selected from the group consisting ofC₁-C₆-alkyl, OH, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and—C(O)O—C₁-C₆-alkyl.

Formula II compounds conform to the following structure:

In Formula II, R^(1B) and R^(2B) are independently selected from thegroup consisting of H, C₁-C₆-alkyl, and C₂-C₆-alkenyl.

R^(3B) and R^(4B) are independently selected from the group consistingof H, C₁-C₆-alkyl, C₂-C₆-alkenyl, and C₁-C₆-alkoxy.

Alternatively, R^(3A) and R^(4A), together with the carbon atoms towhich they are attached, represent a fused 5- to 6-membered heterocycle,wherein 1 to 2 ring members are selected from NR^(1B), O, and S.

Ar^(1B) and Ar^(2B) are independently C₆-C₁₀-aryl.

Any alkyl, alkenyl, alkoxy, aryl, and heteroaryl is optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₆-alkyl, OH, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl,and —C(O)O—C₁-C₆-alkyl.

Formula III compounds conform to the following structure:

In Formula III, R^(1C), R^(2C), and R^(3C) are independently selectedfrom the group consisting of H, C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, CN,C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and —C(O)O—C₁-C₆-alkyl.

Integer f is 0, 1, 2, or 3; and integers o, p, and q are independentlyselected from 0, 1, 2, 3, 4, and 5.

X and Y are independently selected from the group consisting of a bond,—NH—, and —CH₂—.

Cy^(1C) is selected from the group consisting of C₃-C₈-cycloalkyl and 3-to 7-membered heterocyclo wherein 1-3 ring members are selected from N,O, and S.

Any alkyl, alkenyl, alkoxy, aryl, and heterocyclo is optionallysubstituted with 1-3 substituents selected from the group consisting ofC₁-C₆-alkyl, OH, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and—C(O)O—C₁-C₆-alkyl.

Formula IV compounds conform to the following structure:

In Formula IV, Cy^(1D) is a 5- to 6-membered heteroaryl (wherein 1-3ring members are N) or C₆-C₁₀-aryl, wherein Cy^(1D) is optionallysubstituted by 1-3 R^(1D).

Cy^(2D) is a 5- to 6-membered heteroaryl (wherein 1-3 ring members areN) or C₆-C₁₀-aryl, wherein Cy^(2D) is optionally substituted by 1-3R^(2D).

R^(1D) in each instance is independently selected from the groupconsisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, C₆-C₁₀-aryl.

R^(2D) in each instance is independently is selected from the groupconsisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, C₆-C₁₀-aryl.

R^(1D) and R^(2D) are not simultaneously C₆-C₁₀-aryl.

R^(3D) in each instance is independently selected from the groupconsisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, and halo.

Integer r is 0, 1, 2, or 3.

Any alkyl, alkenyl, alkoxy, heteroaryl, and aryl is optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₆-alkyl, OH, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl,and —C(O)O—C₁-C₆-alkyl.

Another embodiment of the disclosure is a compound according to any oneof Formula I-IV for use treating a patient suffering from amesenchymally-derived or mesenchymally-transformed cancer.

The disclosure further provides in another embodiment, optionally incombination with any other embodiment disclosed herein, a method oftreating a subject suffering from a disease or condition that ischaracterized by the expression of vimentin. The method comprisesadministering to the subject a therapeutically effective amount of acompound, or a pharmaceutically acceptable salt thereof, according toany one of Formulae I-IV.

Still another embodiment, optionally in combination with any otherembodiment disclosed herein, is a compound or a pharmaceuticallyacceptable salt thereof, according to any one of Formulae I-IV, for usein treating a patient suffering from a mesenchymally-derived ormesenchymally-transformed cancer, or from a disease or condition that ischaracterized by the expression of vimentin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Relative viability measurements of FOXC2 (lower trace) andcontrol HMLER (upper trace) cells exposed to the indicated doses ofCompound 1 for 72 hours (n=3, mean and s.e.m.).

FIG. 2: Relative proliferation measurements of the indicated VIMnegative and STS cell lines treated with a concentration response ofCompound 1 (n=3, mean and s.e.m.).

FIG. 3: Quantification of mammospheres from an in vitro mammosphereformation assay with the indicated cell types. Pretreatment indicates a72-hour treatment with 1 μM Compound 1 before plating in mammosphereformation conditions. The expression “during assay” indicates 1 μMcompound treatment during the mammosphere assay (14 days) (n=3, mean ands.d.).

FIG. 4: Western blotting analysis of phosphorylated VIM protein contentfrom FOXC2-HMLER cells treated for 24 hours with Compound 1 (500 nM).

FIG. 5: Quantification of immunofluorescent staining for P-S56-VIM fromFOXC2-HMLER cells treated with the indicated doses of Compound 1 (n=3,mean and s.d.; **P<0.005, ***P<0.0005; t-test)

FIG. 6: Representative images of immunofluorescent staining forP-S56-VIM from FOXC2-HMLER cells treated with Compound 1.

FIG. 7: Representative confocal images of thymidine synced FOXC2-HMLERcells at metaphase immunostained for VIM and β-tubulin (TUB).

DETAILED DESCRIPTION Definitions

“Alkyl” refers to straight, branched chain, or cyclic hydrocarbylgroups, e.g., “cycloalkyl,” including from 1 to about 20 carbon atoms.For instance, an alkyl can have from 1 to 10 carbon atoms or 1 to 6carbon atoms. Exemplary alkyl includes straight chain alkyl groups suchas methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, and the like, and also includes branched chainisomers of straight chain alkyl groups, for example without limitation,—CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂, —C(CH₃)₃, —C(CH₂CH₃)₃,—CH₂CH(CH₃)₂, —CH₂CH(CH₃)(CH₂CH₃), —CH₂CH(CH₂CH₃)₂, —CH₂C(CH₃)₃,—CH₂C(CH₂CH₃)₃, —CH(CH₃)CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₃)₂,—CH₂CH₂CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₂CH₃)₂, —CH₂CH₂C(CH₃)₃,—CH₂CH₂C(CH₂CH₃)₃, —CH(CH₃)CH₂CH(CH₃)₂, —CH(CH₃)CH(CH₃)CH(CH₃)₂, and thelike. Thus, alkyl groups include primary alkyl groups, secondary alkylgroups, and tertiary alkyl groups.

The phrase “substituted alkyl” refers to alkyl substituted at one ormore positions, for example, 1, 2, 3, 4, 5, or even 6 positions, whichsubstituents are attached at any available atom to produce a stablecompound, with substitution as described herein. “Optionally substitutedalkyl” refers to alkyl or substituted alkyl.

Each of the terms “halogen,” “halide,” and “halo” refers to —F, —Cl,—Br, or —I.

The term “alkenyl” refers to straight, branched chain, or cyclichydrocarbyl groups, e.g., “cycloalkenyl,” including from 2 to about 20carbon atoms, such as 2 to carbon atoms, having 1-3, 1-2, or at leastone carbon to carbon double bond. The term “cycloalkenyl” refersspecifically to cyclic alkenyl, such as C₃-C₆-cycloalkenyl.

“Substituted alkenyl” refers to alkene substituted at 1 or more, e.g.,1, 2, 3, 4, 5, or even 6 positions, which substituents are attached atany available atom to produce a stable compound, with substitution asdescribed herein. “Optionally substituted alkene” refers to alkene orsubstituted alkene.

The term “alkoxy” refers to an —O-alkyl group having the indicatednumber of carbon atoms. For example, a (C₁-C₆)alkoxy group includes—O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-butyl, —O-sec-butyl,—O-tert-butyl, —O-pentyl, —O-isopentyl, —O-neopentyl, —O-hexyl,—O-isohexyl, and —O-neohexyl.

“Aryl” when used alone or as part of another term means a carbocyclicaromatic group whether or not fused having the number of carbon atomsdesignated or if no number is designated, up to 14 carbon atoms, such asa C₆-C₁₄-aryl. Particular aryl groups are phenyl, naphthyl, biphenyl,phenanthrenyl, naphthacenyl, and the like (see e.g. Lang's Handbook ofChemistry (Dean, J. A., ed) 13^(th) ed. Table 7-2 [1985]). A particulararyl is phenyl. “Aryl” also includes aromatic ring systems that areoptionally fused with a cycloalkyl ring, as herein defined.

A “substituted aryl” is an aryl that is independently substituted withone or more substituents attached at any available atom to produce astable compound, wherein the substituents are as described herein.“Optionally substituted aryl” refers to aryl or substituted aryl.

The term “heteroatom” refers to N, O, and S. Inventive compounds thatcontain N or S atoms can be optionally oxidized to the correspondingN-oxide, sulfoxide, or sulfone compounds.

“Heteroaryl,” alone or in combination with any other moiety describedherein, refers to a monocyclic aromatic ring structure containing 5 to10, such as 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to10 atoms, containing one or more, such as 1-4, 1-3, or 1-2, heteroatomsindependently selected from the group consisting of O, S, and N.Heteroaryl is also intended to include oxidized S or N, such assulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. A carbon orheteroatom is the point of attachment of the heteroaryl ring structuresuch that a stable compound is produced. Examples of heteroaryl groupsinclude, but are not limited to, pyridinyl, pyridazinyl, pyrazinyl,quinaoxalyl, indolizinyl, benzo[b]thienyl, quinazolinyl, purinyl,indolyl, quinolinyl, pyrimidinyl, pyrrolyl, pyrazolyl, oxazolyl,thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl,tetrazolyl, imidazolyl, triazolyl, furanyl, benzofuryl, and indolyl.

A “substituted heteroaryl” is a heteroaryl that is independentlysubstituted, unless indicated otherwise, with one or more, e.g., 1, 2,3, 4 or 5, also 1, 2, or 3 substituents, also 1 substituent, attached atany available atom to produce a stable compound, wherein thesubstituents are as described herein. “Optionally substitutedheteroaryl” refers to heteroaryl or substituted heteroaryl.

“Heterocycloalkyl” or “heterocyclo” means a saturated or unsaturatednon-aromatic monocyclic, bicyclic, tricyclic or polycyclic ring systemthat has from 3 to 14, such as 3 to 6, atoms in which from 1 to 3 carbonatoms in the ring are replaced by heteroatoms of O, S or N. Aheterocycloalkyl is optionally fused with aryl or heteroaryl of 5-6 ringmembers, and includes oxidized S or N, such as sulfinyl, sulfonyl andN-oxide of a tertiary ring nitrogen. The point of attachment of theheterocycloalkyl ring is at a carbon or heteroatom such that a stablering is retained. Examples of heterocycloalkyl groups include withoutlimitation morpholino, tetrahydrofuranyl, dihydropyridinyl, piperidinyl,pyrrolidinyl, piperazinyl, dihydrobenzofuryl, and dihydroindolyl.

“Optionally substituted heterocycloalkyl” denotes heterocycloalkyl thatis substituted with 1 to 3 substituents, e.g., 1, 2 or 3 substituents,attached at any available atom to produce a stable compound, wherein thesubstituents are as described herein.

The term “nitrile” or “cyano” can be used interchangeably and refer to a—CN group which is bound to a carbon atom of a heteroaryl ring, arylring and a heterocycloalkyl ring.

The substituent —CO₂H may be replaced with bioisosteric replacementssuch as:

and the like, wherein R has the same definition as R^(A) as definedherein. See, e.g., THE PRACTICE OF MEDICINAL CHEMISTRY (Academic Press:New York, 1996), at page 203.

Compounds described herein can exist in various isomeric forms,including configurational, geometric, and conformational isomers,including, for example, cis- or trans-conformations. The compounds mayalso exist in one or more tautomeric forms, including both singletautomers and mixtures of tautomers. The term “isomer” is intended toencompass all isomeric forms of a compound of this disclosure, includingtautomeric forms of the compound. The compounds of the presentdisclosure may also exist in open-chain or cyclized forms. In some casesone or more of the cyclized forms may result from the loss of water. Thespecific composition of the open-chain and cyclized forms may bedependent on how the compound is isolated, stored or administered. Forexample, the compound may exist primarily in an open-chained form underacidic conditions but cyclize under neutral conditions. All forms areincluded in the disclosure.

Some compounds described herein can have asymmetric centers andtherefore exist in different enantiomeric and diastereomeric forms. Acompound of the disclosure can be in the form of an optical isomer or adiastereomer. Accordingly, the disclosure encompasses compounds andtheir uses as described herein in the form of their optical isomers,diastereoisomers and mixtures thereof, including a racemic mixture.Optical isomers of the compounds of the disclosure can be obtained byknown techniques such as asymmetric synthesis, chiral chromatography,simulated moving bed technology or via chemical separation ofstereoisomers through the employment of optically active resolvingagents.

Unless otherwise indicated, the term “stereoisomer” means onestereoisomer of a compound that is substantially free of otherstereoisomers of that compound. Thus, a stereomerically pure compoundhaving one chiral center will be substantially free of the oppositeenantiomer of the compound. A stereomerically pure compound having twochiral centers will be substantially free of other diastereomers of thecompound. A typical stereomerically pure compound comprises greater thanabout 80% by weight of one stereoisomer of the compound and less thanabout 20% by weight of other stereoisomers of the compound, for examplegreater than about 90% by weight of one stereoisomer of the compound andless than about 10% by weight of the other stereoisomers of thecompound, or greater than about 95% by weight of one stereoisomer of thecompound and less than about 5% by weight of the other stereoisomers ofthe compound, or greater than about 97% by weight of one stereoisomer ofthe compound and less than about 3% by weight of the other stereoisomersof the compound.

If there is a discrepancy between a depicted structure and a name givento that structure, then the depicted structure controls. Additionally,if the stereochemistry of a structure or a portion of a structure is notindicated with, for example, bold or dashed lines, the structure orportion of the structure is to be interpreted as encompassing allstereoisomers of it. In some cases, however, where more than one chiralcenter exists, the structures and names may be represented as singleenantiomers to help describe the relative stereochemistry. Those skilledin the art of organic synthesis will know if the compounds are preparedas single enantiomers from the methods used to prepare them.

In this description, a “pharmaceutically acceptable salt” is apharmaceutically acceptable, organic or inorganic acid or base salt of acompound of the disclosure. Representative pharmaceutically acceptablesalts include, e.g., alkali metal salts, alkali earth salts, ammoniumsalts, water-soluble and water-insoluble salts, such as the acetate,amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate,benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide,butyrate, calcium, calcium edetate, camsylate, carbonate, chloride,citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate,esylate, fiunarate, gluceptate, gluconate, glutamate,glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate,lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate,oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate,einbonate), pantothenate, phosphate/diphosphate, picrate,polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate,subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate,tartrate, teoclate, tosylate, triethiodide, and valerate salts. Apharmaceutically acceptable salt can have more than one charged atom inits structure. In this instance the pharmaceutically acceptable salt canhave multiple counterions. Thus, a pharmaceutically acceptable salt canhave one or more charged atoms and/or one or more counterions.

The terms “treat”, “treating” and “treatment” refer to the ameliorationor eradication of a disease or symptoms associated with a disease. Incertain embodiments, such terms refer to minimizing the spread orworsening of the disease resulting from the administration of one ormore prophylactic or therapeutic agents to a patient with such adisease.

The terms “prevent,” “preventing,” and “prevention” refer to theprevention of the onset, recurrence, or spread of the disease in apatient resulting from the administration of a prophylactic ortherapeutic agent.

The term “effective amount” refers to an amount of a compound of thedisclosure or other active ingredient sufficient to provide atherapeutic or prophylactic benefit in the treatment or prevention of adisease or to delay or minimize symptoms associated with a disease.Further, a therapeutically effective amount with respect to a compoundof the disclosure means that amount of therapeutic agent alone, or incombination with other therapies, that provides a therapeutic benefit inthe treatment or prevention of a disease. Used in connection with acompound of the disclosure, the term can encompass an amount thatimproves overall therapy, reduces or avoids symptoms or causes ofdisease, or enhances the therapeutic efficacy of or synergies withanother therapeutic agent.

A “patient” or subject” includes an animal, such as a human, cow, horse,sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbitor guinea pig. The animal can be a mammal such as a non-primate and aprimate (e.g., monkey and human). In one embodiment, a patient is ahuman, such as a human infant, child, adolescent or adult.

Compounds

As described generally above, the present disclosure provides in someembodiments compounds and pharmaceutically acceptable salts thereof,wherein the compounds conform to Formula (I):

R^(1A) in each instance is independently selected from the groupconsisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, and C₁-C₆-alkoxy, halo, CN,—S—C₁-C₆-alkyl, —C(O)N(R^(2A))₂.

R^(2A) in each instance is independently selected from H andC₁-C₆-alkyl;

Each ---, if present, represent single bonds; and n is 0, 1, 2, or 3.

Het^(A) is 6-membered monocyclic or 9- to 10-membered bicyclicheteroaryl, wherein 1 to 3 ring members are N.

Het^(A) and any alkyl, alkenyl, alkoxy, and aryl is optionallysubstituted with 1-3 substituents selected from the group consisting ofC₁-C₆-alkyl, halo, OH, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and—C(O)O—C₁-C₆-alkyl.

In some embodiments, each --- is absent and Het^(A) is a 9- to10-membered bicyclic heteroaryl. Exemplary bicyclic heteroaryl groupsinclude but are not limited to indole, isoindole, indolizine, quinolone,isoquinoline, quinolizine, indazole, quinazoline, cinnoline,quinoxaline, and phthalazine.

Other embodiments provide for substitution of Het^(A). For example,Het^(A) is substituted by 1-3 halogens, such as Cl and F.

In other embodiments, optionally in combination with any otherembodiment, R^(1A) is a halogen, and n is 1, 2, or 3.

Specific examples of Formula I compounds are presented in Table 1 as setforth below.

Formula II compounds are represented by the structure:

R^(1B) and R^(2B) are independently selected from the group consistingof H, C₁-C₆-alkyl, and C₂-C₆-alkenyl.

R^(3B) and R^(4B) are independently selected from the group consistingof H, C₁-C₆-alkyl, C₂-C₆-alkenyl, and C₁-C₆-alkoxy.

Alternatively, R^(3A) and R^(4A), together with the carbon atoms towhich they are attached, represent a fused 5- to 6-membered heterocycle,wherein 1 to 2 ring members are selected from NR^(1B), O, and S.

Ar^(1B) and Ar^(2B) are independently C₆-C₁₀-aryl.

Any alkyl, alkenyl, alkoxy, aryl, and heteroaryl is optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₆-alkyl, OH, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl,and —C(O)O—C₁-C₆-alkyl.

In some embodiments, Ar^(1B) and Ar^(2B) are independently andoptionally substituted phenyl. Exemplary substituents in this contextinclude C₁-C₆-alkyl and C₁-C₆-alkoxy.

Optionally in combination with other embodiments, another embodimentprovides for each of R^(1B) and R^(2B) as H.

Specific examples of Formula II compounds are presented in Table 2 asset forth below.

Formula III compounds are represented by the structure:

R^(1C), R^(2C), and R^(3C) are independently selected from the groupconsisting of H, C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, CN, C₁-C₆-alkoxy,—C(O)C₁-C₆-alkyl, and —C(O)O—C₁-C₆-alkyl.

The integer f is 0, 1, 2, or 3; o, p, and q are independently selectedfrom 0, 1, 2, 3, 4, and 5.

X and Y are independently selected from the group consisting of a bond,—NH—, and —CH₂—.

Cy^(1C) is selected from the group consisting of C₃-C₈-cycloalkyl and 3-to 7-membered hetercyclo wherein 1-3 ring members are selected from N,O, and S.

Any alkyl, alkenyl, alkoxy, aryl, and heterocyclo is optionallysubstituted with 1-3 substituents selected from the group consisting ofC₁-C₆-alkyl, OH, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and—C(O)O—C₁-C₆-alkyl;

According to some embodiments, X and Y each is a bond; f is 0; andCy^(1C) is an optionally substituted 3- to 7-membered hetercyclo wherein1-3 ring members are N.

Other Formula III compounds, according to various embodiments, featureCy^(1C) as a 6-membered hetercyclo wherein 1-2 ring members are N.Examples of such a heterocyclo include piperidine, hexahydro-pyrimidine,and piperazine.

Specific examples of Formula III compounds are presented in Table 3 asset forth below.

Formula IV compounds are represented by the structure:

Cy^(1D) is a 5- to 6-membered heteroaryl (wherein 1-3 ring members areN) or C₆-C₁₀-aryl, wherein Cy^(1D) is optionally substituted by 1-3R^(1D).

Cy^(2D) is a 5- to 6-membered heteroaryl (wherein 1-3 ring members areN) or C₆-C₁₀-aryl, wherein Cy^(2D) is optionally substituted by 1-3R^(2D).

R^(1D) in each instance is independently selected from the groupconsisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, C₆-C₁₀-aryl.

R^(2D) in each instance is independently is selected from the groupconsisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, C₆-C₁₀-aryl.

R^(1D) and R^(2D) are not simultaneously C₆-C₁₀-aryl.

R^(3D) in each instance is independently selected from the groupconsisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, and halo.

Integer r is 0, 1, 2, or 3.

Any alkyl, alkenyl, alkoxy, heteroaryl, and aryl is optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₆-alkyl, OH, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl,and —C(O)O—C₁-C₆-alkyl.

In some embodiments, Cy^(1D) is an optionally substituted 5- to6-membered heteroaryl (wherein 1-3 ring members are N); and Cy^(2D) isan optionally substituted 5- to 6-membered heteroaryl (wherein 1-3 ringmembers are N). Examples of 6-membered heteroaryl are described above.Examples of 5-membered heteroaryl include pyrrole, imidazole, andpyrazole.

In other embodiments, optionally in combination with any otherembodiment, Cy^(1D) is substituted by one R^(1D) that itself isoptionally substituted C₆-C₁₀-aryl. Alternatively, Cy^(2D) issubstituted by one R^(2D) that is optionally substituted C₆-C₁₀-aryl.Exemplary aryl groups include phenyl and naphthyl.

Specific examples of Formula IV compounds are presented in Table 4 asset forth below.

Pharmaceutical Composition

The disclosure also provides a pharmaceutical composition comprising atherapeutically effective amount of one or more compounds according toany of Formula I-VI or a pharmaceutically acceptable salt, solvate,stereoisomer, tautomer, or prodrug, in admixture with a pharmaceuticallyacceptable carrier. In some embodiments, the composition furthercontains, in accordance with accepted practices of pharmaceuticalcompounding, one or more additional therapeutic agents, pharmaceuticallyacceptable excipients, diluents, adjuvants, stabilizers, emulsifiers,preservatives, colorants, buffers, flavor imparting agents.

In one embodiment, the pharmaceutical composition comprises a compoundselected from those illustrated in Tables 1-4 or a pharmaceuticallyacceptable salt, solvate, stereoisomer, tautomer, or prodrug thereof,and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present disclosure is formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular subject being treated,the clinical condition of the subject, the cause of the disorder, thesite of delivery of the agent, the method of administration, thescheduling of administration, and other factors known to medicalpractitioners.

The “therapeutically effective amount” of the compound that isadministered is governed by such considerations, and is the minimumamount necessary to exert a cytotoxic effect on a mesenchymally-derivedor mesenchymally-transformed cancer. Such amount may be below the amountthat is toxic to normal cells, or the subject as a whole. Generally, theinitial therapeutically effective amount of the compound of the presentdisclosure that is administered parenterally per dose is in the range ofabout 0.01-2000 mg/kg, for example about 0.01 to about 200 mg/kg, about0.1 to 20 mg/kg of patient body weight per day, with the typical initialrange of compound used being 0.3 to 15 mg/kg/day. Oral unit dosageforms, such as tablets and capsules, may contain from about 25 to about200 mg of the compound of the present disclosure.

The inventive compositions can be administered orally, topically,parenterally, by inhalation or spray or rectally in dosage unitformulations. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection, orinfusion techniques.

Suitable oral compositions in accordance with the disclosure includewithout limitation tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsion, hard or softcapsules, syrups or elixirs.

Encompassed within the scope of the disclosure are pharmaceuticalcompositions suitable for single unit dosages that comprise a compoundof the disclosure or its pharmaceutically acceptable stereoisomer,prodrug, salt, solvate, hydrate, or tautomer and a pharmaceuticallyacceptable carrier.

Inventive compositions suitable for oral use may be prepared accordingto any method known to the art for the manufacture of pharmaceuticalcompositions. For instance, liquid formulations of the present compoundscontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations of the arginase inhibitor.

For tablet compositions, the compound in admixture with non-toxicpharmaceutically acceptable excipients is used for the manufacture oftablets. Examples of such excipients include without limitation inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known coating techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby to provide asustained therapeutic action over a desired time period. For example, atime delay material such as glyceryl monostearate or glyceryl distearatemay be employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

For aqueous suspensions the inventive compound is admixed withexcipients suitable for maintaining a stable suspension. Examples ofsuch excipients include without limitation are sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.

Oral suspensions can also contain dispersing or wetting agents, such asnaturally-occurring phosphatide, for example, lecithin, or condensationproducts of an alkylene oxide with fatty acids, for examplepolyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohols, for example,heptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol.

Sweetening agents such as those set forth above, and flavoring agentsmay be added to provide palatable oral preparations. These compositionsmay be preserved by the addition of an anti-oxidant such as ascorbicacid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions of the disclosure may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monoleate, and condensaturatedionproducts of the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monoleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative, and flavoring and coloringagents. The pharmaceutical compositions may be in the form of a sterileinjectable, an aqueous suspension or an oleaginous suspension. Thissuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents which havebeen mentioned above. The sterile injectable preparation may also besterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The compounds of Formulae I-VI may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. Such materials are cocoa butter and polyethylene glycols.

Compositions for parenteral administrations are administered in asterile medium. Depending on the vehicle used and concentration theconcentration of the drug in the formulation, the parenteral formulationcan either be a suspension or a solution containing dissolved drug.Adjuvants such as local anesthetics, preservatives and buffering agentscan also be added to parenteral compositions.

Methods of Use

Cancer cells that are derived from mesenchymal tissues or that areinduced to adopt a cancer stem cell-like mesenchymal state have beendemonstrated to be largely resistant to standard chemotherapies,necessitating the identification of new effective treatment strategies.Demonstrated by the appended examples, the Formula I-IV compoundsirreversibly inhibit the growth of mesenchymally transformed cancercells.

Accordingly, in an embodiment of present disclosure there is provided amethod for treating a patient cancer suffering from amesenchymally-derived or mesenchymally-transformed cancer. The methodcomprises administering to the subject a therapeutically effectiveamount of a compound, or a pharmaceutically acceptable salt thereof,according to any one of Formulae I-IV as described herein.

In some embodiments, the cancer is a breast cancer. Various breastcancers include ductal carcinoma in situ, invasive ductal carcinoma,lobular carcinoma. For example, the present methods are particularlyeffective inhibiting triple-negative breast cancer.

In other embodiments, the cancer is a sarcoma. Illustrative sarcomasinclude Askin's tumor, sarcoma botryoides, chondrosarcoma, Ewing'ssarcoma, malignant hemangioendothelioma, malignant Schwannoma, andosteosarcoma. The sarcoma also can be a soft tissue sarcoma, includingalveolar soft part sarcoma, angiosarcoma, cystosarcoma Phyllodes,dermatofibrosarcoma protuberans, desmoid tumor, desmoplastic small roundcell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma,extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromaltumor, hemangiopericytoma (“solitary fibrous tumor”), angiosarcoma,Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma,Rhabdomyosarcoma, synovial sarcoma, and undifferentiated pleomorphicsarcoma.

Another embodiment provides a method for selectively inhibitingFOXC2-expressing breast cancer stem cells. The method comprisescontacting the cells with an effective amount of a compound, or apharmaceutically acceptable salt thereof, according to any one ofFormulae I-IV as described herein. The contacting occurs in vivo.Alternatively, in accordance with an embodiment, the contacting occursin vitro or ex vivo.

Compounds of the present disclosure chemically target the intermediatefilament protein, vimentin, and the compounds therefore promote mitoticcatastrophe, such as in mesenchymally-derived ormesenchymally-transformed cancers. The compounds also are useful intreating diseases or conditions that are characterized by the expressionof vimentin (K. M. Ridge et al., Methods Enzymol. 568 (2016) 389-426).The diseases and conditions include inflammatory diseases (G. Dos Santoset al., Nature Communications 6 (2015) 6574) such as Crohn's disease (C.Stevens et al., Gut 62(5) (20123) 695-707), cancers such as lung cancer(M. E. Kidd et al., American Journal of Respiratory Cell and MolecularBiology 50(1) (2014) 1-6), congenital cataracts (M. Muller et al., HumanMolecular Genetics 18(6) (2009) 1052-1057), and neuropathies such asgiant axonal neuropathy (GAN) (S. Mahammad et al., Journal of ClinicalInvestigation 123(5) (2013) 1964-1975).

EXAMPLES

The present disclosure will be additionally understood by reference tothe following examples. The examples should not, however, be construedas limiting the scope of the present disclosure.

Example 1: High Throughput Screen Identifies Selective Inhibitors ofFOXC2-Expressing Cells

An engineered cancer cell line HMLER as an isogenic screening system wasemployed to identify compounds that are selectively toxic to the FOXC2positive genotype. HMLER cells are derived from mammary epithelial cells(HMECs), but are virally transformed with the K-Ras oncogene (G12V),human telomerase reverse transcriptase (hTERT), and SV40 large-T antigen(B. Elenbaas, B. et al. Genes Dev. 15 (2001) 50-65). Expression of thesegenes endows HMLER cells with functional immortality and tumorigenicpotential at high seeding number (>10⁶ cells; Hollier (2013)). HMLERcells retain epithelial characteristics and sensitivity to variousEMT-inducing stimuli (e.g. TGF-β1) and have historically been used toinvestigate the effects of single EMT related genes on cancer stemness(Mani (2008)). Further, retroviral expression of FOXC2 endows thesecells with metastatic potential and the properties of stem cells,including the ability to form mammospheres, resist conventionalchemotherapeutic agents, and form tumors at limiting dilutions (10³cells; Hollier (2013)). This example thus employed FOXC2-expressingHMLER cells (designated FOXC2-HMLER throughout) that were generatedusing retroviral transgene delivery, with the isogenic HMLER cell lineas a control (Hollier (2013)).

Cell Lines:

The propagation conditions and generation of FOXC2-HMLER, HMLER,SNAIL-HMLER, SNAIL-HMLE, and SUM159 cells have been described previously(Mani (2008)). MCF-7, HepG2, HCT116, and MDA-MB-231 cells were from ATCCand maintained in DMEM medium (Corning) supplemented with 10% FBS(Corning) with Anti-anti (Gibco). The soft tissue sarcoma cell linepanel was purchased from ATCC (TCP-1019) and maintained in therecommended medium for each cell type. HUVEC cells (pooled donor) weremaintained in EBM-2 medium (both from Lonza Walkersville). Primary humanlung fibroblasts were maintained in Fibroblast Medium (both fromSciencell Research Laboratories). For cell growth experiments, HLFs weregrown in DMEM supplemented with 2% FBS and Anti-anti. For allexperiments, primary human cells were used at a passage no greater than4.

High Throughput Screening and Miniaturized Cell Viability Experiments:

For high throughput screening, FOXC2-HMLER and HMLER cells were platedat 10³ cells per well in white 384-well plates in 50 μL of MEGM medium(Lonza). Cells were allowed to attach for one hour before compound wastransferred to each well as a DMSO solution using a 100 nL pintool headaffixed to a PerkinElmer FX instrument. After 72-hour incubation, 30 μLof a Cell Titer Glo solution (diluted 1:6 in water, Promega) wasdispensed into each well and luminance values were recorded using anEnvision plate reader. Compounds which decreased viability ofFOXC2-HMLER cells three Z scores below plate mean but did not decreasecontrol HMLER viability more than one Z score below plate mean weredeemed primary screening hits. Selected hit compounds were reorderedfrom ChemDiv and tested for selective toxicity in 10-point responseassays as above. All other miniaturized, selective viability experimentswere performed as above using the indicated growth medium.

More specifically, in the high throughput screening assay above, eachcell line was plated at 1,000 cells per well in 384-well plates, treatedfor 72 hours with 2 μM of test compound, and the viability of each linewas determined in parallel by Cell Titer Glo (Promega) luminancemeasurements. The screen employed a library of ˜50,000 diverseheterocyclic compounds and biologically active small molecules (ChemDiv,San Diego, Calif., USA) for cytotoxic activity against both cell lines.

Preliminary screening of the compounds revealed that FOXC2-HMLER cellswere largely resistant to most chemotherapies tested. For comparison,the commonly used chemotherapy drug doxorubicin (Fisher Scientific)displays modest preferential toxicity to control HMLER cells (IC₅₀ 267nM) relative to FOXC2-HMLER cells (IC₅₀ 447 nM). These results wereconsistent with the notion that these cells represent a cancer stem cellpopulation.

This screening campaign led to the identification of Formulae I-IVcompounds that selectively inhibit the growth of FOXC2-expressing HMLERcells at sub-micromolar doses while displaying minimal toxicity tovector control HMLER cells or to non-transformed human mammaryepithelial cells (HMECs) at doses up to 20 μM (see FIG. 1).

Results of the screen are presented in Tables 1 to 4 below,corresponding to compounds of Formulae I-IV, respectively. Activities ofthe compounds are scored in relation to the activities of “parent”compounds in each table, i.e., those labeled as compounds 1, 2, 3, and4, as follows:

Activity score Activity +++ More potent than parent ++ IC₅₀ < 1 μM;similar activity to parent + 20 μM > IC₅₀ > 1 μM; less potent thanparent − IC₅₀ > 20 μM; no detectable activity

TABLE 1 Inhibition of FOXC2-expressing HMLER cells by Formula ICompounds Compound Activity No. Structure Score 1

++ 1A

+ 1B

+ 1C

+ 1D

+ 1E

− 1F

− 1G

− 1SRH

+++ 1SRI

+ 1SRJ

+ 1SRK

+ 1SRL

++ 1SRM

++ 1SRN

++ 1SRO

+ 1SRP

+ 1SRQ

+ 1SRU

+ 1SRT

++ 1SRU

++ 1SRV

+ 1SRW

+ 1SRX

− 1SRY

− 1SRZ

− 1SRAA

+ 1SRAB

+ 1SRAC

+ 1SRAD

+ 1SRAE

− 1SRAF

− 1SRAG

++ 1SRAH

+++ 1SRAI

++ 1SRAJ

+ 1SRAK

++ 1SRAL

+ 1SRAM

− 1SRAN

++

TABLE 2 Inhibition of FOXC2-expressing HMLER cells by Formula IICompounds Compound Activity No. Structure Score 2A

+++ 2

++ 2B

++ 2C

++ 2D

+ 2E

+ 2F

+ 2G

+ 2H

+ 2I

+ 2J

+ 2K

+ 2L

+ 2M

+ 2N

+ 2O

+ 2P

+ 2Q

+ 2R

+ 2S

+ 2T

+ 2U

+ 2V

+ 2W

+ 2X

+ 2Y

+ 2Z

+ 2AA

+ 2AB

+ 2AC

+ 2AD

+ 2AE

+ 2AF

+ 2AG

+ 2AH

+ 2AI

+ 2AJ

+ 2AK

+ 2AL

+ 2AM

+ 2AN

+ 2AO

+ 2AP

+ 2AQ

− 2AR

− 2AS

− 2AT

− 2AU

− 2AV

− 2AW

− 2AX

− 2AY

− 2AZ

− 2BA

− 2BB

− 2BC

− 2BD

− 2BE

− 2BF

− 2BG

− 2BH

− 2BI

− 2BJ

− 2BK

− 2BL

− 2BM

− 2BN

− 2BO

− 2BP

− 2BQ

− 2BR

− 2BS

− 2BT

− 2BU

− 2BV

− 2BW

− 2BX

− 2BY

− 2BZ

− 2CA

− 2CB

− 2CC

− 2CD

− 2CE

− 2CF

− 2CG

− 2CH

− 2CI

− 2CJ

− 2CK

− 2CL

− 2CM

− 2CN

− 2CO

− 2CP

− 2CQ

− 2CR

− 2CS

− 2CT

− 2CU

− 2CV

− 2CW

− 2CX

− 2CY

− 2CZ

− 2DA

− 2DB

− 2DC

− 2DD

− 2DE

− 2DF

− 2DG

− 2DH

− 2DI

− 2DJ

− 2DK

− 2DL

− 2DM

− 2DN

− 2DO

− 2DP

− 2DQ

− 2DR

− 2DS

− 2DT

− 2DU

− 2DV

− 2DW

− 2DX

− 2DY

− 2DZ

− 2EA

− 2EB

− 2EC

− 2ED

− 2EE

− 2EF

− 2EG

− 2EH

− 2EI

− 2EJ

− 2EK

− 2EL

− 2EM

− 2EN

− 2EO

− 2EP

− 2EQ

− 2ER

− 2ES

− 2ET

− 2EU

− 2EV

− 2EW

− 2EX

− 2EY

− 2EZ

− 2FA

− 2FB

− 2FC

− 2FD

− 2FE

− 2FF

− 2FG

− 2FH

− 2FI

− 2FJ

− 2FK

− 2FL

− 2FM

− 2FN

− 2FO

− 2FP

− 2FQ

− 2FR

− 2FS

− 2FT

− 2FU

− 2FV

− 2FW

− 2FX

− 2FY

− 2FZ

− 2GA

− 2GB

− 2GC

− 2GD

− 2GE

− 2GF

− 2GG

− 2GH

− 2GI

− 2GJ

− 2GK

− 2GL

− 2GM

− 2GN

− 2GO

− 2GP

− 2GQ

− 2GR

− 2GS

− 2GT

− 2GU

− 2GV

− 2GW

− 2GX

− 2GY

− 2GZ

− 2HA

− 2HB

− 2HC

− 2HD

− 2HE

− 2HF

− 2HG

− 2HH

− 2HI

− 2HJ

− 2HK

− 2HL

− 2HM

− 2HN

− 2HO

− 2HP

− 2HQ

− 2HR

− 2HS

− 2HT

− 2HU

− 2HV

− 2HW

− 2HX

− 2HY

− 2HZ

− 2IA

− 2IB

− 2IC

− 2ID

− 2IE

− 2IF

− 2IG

− 2IH

− 2II

− 2IJ

− 2IK

− 2IL

− 2IM

−

TABLE 3 Inhibition of FOXC2-expressing HMLER cells by Formula IIICompounds Compound Activity No. Structure Score 3A

+++ 3B

+++ 3C

++ 3D

++ 3E

++ 3F

++ 3

++ 3G

+ 3H

+ 3I

+ 3J

+ 3K

+ 3L

+ 3M

+ 3N

+ 3O

+ 3P

+ 3Q

+ 3R

+ 3S

+ 3T

+ 3U

+ 3V

+ 3W

+ 3X

+ 3Y

+ 3Z

+ 3AA

+ 3AB

+ 3AC

+ 3AD

+ 3AE

+ 3AF

+ 3AG

+ 3AG

+ 3AH

+ 3AI

+ 3AJ

+ 3AK

+ 3AL

− 3AM

− 3AN

− 3AO

− 3AP

− 3AQ

− 3AR

− 3AS

− 3AT

− 3AU

− 3AV

− 3AW

− 3AX

− 3AY

− 3AZ

− 3BA

− 3BB

− 3BC

− 3BD

− 3BE

− 3BF

− 3BG

− 3BH

− 3BI

− 3BJ

− 3BK

− 3BL

− 3BM

− 3BN

− 3BO

− 3BP

− 3BQ

− 3BR

− 3BS

− 3BT

− 3BU

− 3BV

− 3BW

− 3BX

− 3BY

− 3BZ

− 3CA

− 3CB

− 3CC

− 3CD

− 3CE

− 3CF

− 3CG

− 3CH

− 3CI

− 3CJ

− 3CK

−

TABLE 4 Inhibition of FOXC2-expressing HMLER cells by Formula IVCompounds Compound Activity No. Structures Score 4A

+++ 4B

+++ 4C

+++ 4D

++ 4E

++ 4

++ 4F

++ 4G

++ 4H

++ 4I

+ 4J

+ 4K

+ 4L

+ 4M

+ 4N

− 4O

− 4P

− 4Q

− 4R

− 4S

− 4T

− 4U

− 4V

− 4W

− 4X

− 4Y

− 4Z

− 4AA

− 4AB

− 4AC

− 4AD

− 4AE

− 4AF

− 4AG

− 4AH

− 4AI

− 4AJ

− 4AK

− 4AL

− 4AM

− 4AN

− 4AO

− 4AP

− 4AQ

− 4AR

− 4AS

− 4AT

− 4AU

−

Example 2: Inhibition of Additional Breast Cancer Cell Lines

Compound 1 was chosen for further study because it exhibited selectivecytotoxicity (FOXC2-HMLER IC₅₀ 234 nM; HMLER IC₅₀>20 μM). In particular,Compound 1 was found to inhibit the growth of three additionalmesenchymal breast cancer cell lines, SNAIL-HMLE, MDA-MB-231, andSUM159, which express FOXC2 from its endogenous locus.

Example 3: Inhibition of Sarcoma Cell Lines

The anti-proliferative activity of Compound 1 was assessed against aselection of soft tissue sarcomas (STS), which collectively refers to abroad grouping of over 50 subtypes of connective tissue derived cancersfor which targeted therapies are lacking (Zambo (2014)). STS tumorsarise from tissues of mesenchymal origin and, by default, they expressthe intermediate filament and mesenchymal marker vimentin VIMirrespective of subtype (G. Lahat, G. et al. PLoS One 5 (2010) e10105).

A panel of 6 STS cell lines were evaluated, which cell lines includedthe histological subtypes of fibrosarcoma (HT 1080, SW684),rhabdomyosarcoma (RD), fibrous histiocytoma (GCT), liposarcoma (SW872),and synovial sarcoma (SW982). All STS cell lines were first confirmed toexpress vimentin (VIM) at similar levels to FOXC2-HMLER cells by Westernblotting analysis.

Western blotting was performed essentially as described previously (M.J. Bollong et al. ACS Chem. Biol. 10 (2015) 2193-2198). Cells werecollected by brief trypsinization and centrifugation at 500 g for 5minutes. Cells were lysed by the addition of RIPA buffer with proteaseand phosphatase inhibitors (Roche). Lysates were incubated on ice for 30minutes before eliminating insoluble protein content by centrifugationat 12,000 g for 5 minutes at 4° C. Protein concentrations weredetermined from absorbance values obtained by a Nanodrop instrument.Equal amounts of protein were then mixed with 2× loading buffer (100 mMTRIS-HCl, 1% SDS, 10% glycerol, 0.1% bromophenol blue10%-mercaptoethanol) and exposed to 95° C. for 5 minutes. Protein wasseparated by SDS-PAGE using 4-12% Bis-Tris Gels (Invitrogen) and thentransferred to PVDF membranes (Invitrogen) using semi-dry transfer.Membranes were blocked for 1 hour at room temperature in 5% non-fat drymilk in TBST (Tris buffered saline with 0.1% Tween 20). Membranes wereincubated overnight with primary antibodies in blocking buffer overnightat the dilutions ranging from 1:500 to 1:2000. After 3 washes with TBST,membranes were then exposed to HRP-conjugated secondary antibodies(1:5000 in blocking buffer, Sigma) for one hour followed by one hour ofadditional washing with TBST. Relative protein content was visualizedusing film and SuperSignal West Dura Substrate. For VIM degradationblots, 20-60 μg of protein content was loaded per gel lane and theanti-VIM antibody V9 (ab8069) was used to blot for VIM protein content.Phospho-protein blots were performed as above with the exception thatprimary antibodies were incubated in 5% bovine serum albumin in TBSTinstead of milk.

Compound 1 was found to effectively inhibit the growth of all cell lineswith similar IC₅₀ values (0.44-1.31 μM; see FIG. 2). Compound 1additionally promoted a robust multinucleation phenotype in all STS celllines over a 24-hour treatment period. In addition, SW872 cells, whichis a liposarcoma cell line that displayed a significant degree of basalmultinucleation, were most sensitive to Compound 1 treatment (IC₅₀ 0.44μM), indicating that existing mutations promoting genomic instabilityare able to serve as predictive markers of sensitivity to compounds ofthis disclosure.

Example 4: Inhibition of Cancer Cell Stemness

Epithelial-mesenchymal transition (EMT) induction endows breast cancercells with the ability to grow as mammospheres in culture (Mani (2008)).This assay principle is based on studies by Dontu demonstrating thatonly undifferentiated mammary stem cells can survive in suspensionculture whereas differentiated mammary epithelium die as a consequenceof anoikis (G. Dontu et al. Genes Dev. 17 (2003) 1253-1270).

Treatment with Compound 1 inhibited the ability of the FOXC2-expressinglines FOXC2-HMLER, SNAIL-HMLER, SUM159, and MDA-MB-231 cells to formmammospheres in culture (FIG. 3). In addition, pretreating these celllines 72 h before plating resulted in similar levels of growthinhibition when compared to conditions in which Compound 1 was presentthroughout the duration of the assay (14 days), further confirming thatCompound 1 irreversibly inhibits the stemness potential of these celltypes (FIG. 3).

Example 5: Synthesis of Photo-Activatable Affinity Probe (PAP) Molecule

A photo-activatable affinity probe (PAP) molecule was synthesized fortarget identification experiments.

All non-aqueous reactions were carried out in oven-dried glassware underan atmosphere of nitrogen. All solvents, starting materials and reagentswere purchased from commercial vendors and used without furtherpurification. All reagent grade solvents used for chromatography werepurchased from Fisher Scientific. A Biotage FLASH column chromatographysystem was used to purify mixtures and the flash column chromatographysilica cartridges were obtained from Biotage. All NMR spectra wererecorded on a Varian INOVA-400 spectrometer. Chemical shifts (δ) arereported in parts per million relative to the residual solvent peak, andcoupling constants (J) are reported in hertz (Hz). HPLC Gradientconditions: solvent A (0.05% TFA in water) and solvent B (0.05% TFA inAcetonitrile): 0-2 min 95% A, 2-12 min 5-95% B (linear gradient), 12-15min 100% B. Detection by UV-Vis (220-400 nm).

3-chloro-2-nitrobenzoyl chloride

To a solution of 2-nitro-3-chlorobenzoic acid (10.08 g, 50 mmol) indichloromethane (67 mL) was added thionyl chloride (18.2 mL, 250 mmol)followed by DMF (3 drops). The reaction was refluxed 2 hours andevaporated in vacuo to give the acid chloride (10.34 g, 94%). 1H-NMR(400 MHz; DMSO-d6): δ 8.01-7.97 (m, 2H), 7.71 (t, J=8.0 Hz, 1H).

1-(3-chloro-2-nitrophenyl)ethan-1-one

To a suspension of magnesium (10 g, 0.41 mol) in dry THF (750 mL) at 50°C. was added ethanol (40.1 mL) and carbon tetrachloride (1 mL). After 30min. a solution of diethyl malonate (62.5 mL, 0.41 mol) in ethanol (28mL, 0.69 mol) was added and the reaction was allowed to stir at 60° C.for 2 hrs. Quench with 10% sulfuric acid (250 mL). Separate andevaporate the organic layer to give a yellow oil. To the oil was addedacetic acid (365 mL), sulfuric acid (49 mL) and water (248 mL). Thismixture was refluxed 10 hours and the product was extracted with ethylacetate. The combined extracts were washed with water followed by brineand dried over sodium sulfate and evaporated in vacuo to give theproduct 65 g, 79%). 1H-NMR (400 MHz; CDCl3): δ 7.76 (d, J=7.8 Hz, 1H),7.70 (d, J=8.1 Hz, 1H), 7.56 (t, J=8.0 Hz, 1H), 2.61 (s, 3H).

1-(2-amino-3-chlorophenyl)ethan-1-one

To a solution of 1-(3-chloro-2-nitrophenyl)ethan-1-one (65 g, 0.33 mol)in glacial acetic acid (500 mL) was added iron powder (55 g, 0.98 mol).Shake the slurry until it thick. Use cooling to prevent a reflux. Letthe mixture sit for 1 hr at 80° C. shaking ever hour to make sure it ismixed. Cool and quench with 10% sodium hydroxide. Extract product byshaking with ethyl acetate and then separating the emulation bycentrifugation followed by decanting the organic layer. Repeat 3 times.Filter through celite, dry over sodium sulfate and evaporate in vacuo togive the aniline (44.1 g, 80%). 1H-NMR (400 MHz; CDCl3): δ 7.76 (d,J=7.8 Hz, 1H), 7.70 (d, J=8.1 Hz, 1H), 7.56 (t, J=8.0 Hz, 1H), 2.61 (s,3H).

8-chlorocinnolin-4-ol

To a suspension of 1-(2-amino-3-chlorophenyl)ethan-1-one (5 g, 29.5mmol) in water (29 mL) at 0° C. was added conc. HCl (205 mL). A solutionof sodium nitrite (2.05 g, 29.7 mmol) in water (7.5 mL) was addeddropwise. The reaction was stirred for 1 hr. at 0° C. then heated at 65°C. for 4 hr. After cooling, the product was collected by filtration. Theproduct was triturated with acetone and recrystallized from boiling 6MHCl to give the product as pink needles (2.81 g, 15.7 mmol). 1H-NMR (400MHz; CDCl3): δ 10.32 (s, 1H), 8.19 (dd, J=8.2, 0.6 Hz, 1H), 7.89 (s,1H), 7.77 (dd, J=7.7, 1.3 Hz, 1H), 7.35 (t, J=7.9 Hz, 1H).

4-bromo-8-chlorocinnoline

A bottle of phosphorus oxybromide (25 g, 87.2 mmol) was melted with aheat gun and poured into chloroform (150 mL). To this solution was added8-chlorocinnolin-4-ol (2.8 g, 15.5 mmol) and was stirred until a thickslurry was obtained (˜20 min). The reaction was refluxed 2 hr. and thenbasified with 10% sodium carbonate. The mixture was filtered throughcelite and extracted with chloroform. The combined organic layers weredried over sodium sulfate and evaporated to give the product (3.5 g,92%) as a green-brown solid. 1H-NMR (400 MHz; DMSO-d6): δ 9.81 (s, 1H),8.23 (d, J=7.5 Hz, 1H), 8.11 (d, J=8.5 Hz, 1H), 7.98 (t, J=8.0 Hz, 1H).

4-chloro-8-chlorocinnoline

To a flask containing 8-chlorocinnolin-4-ol (4.67 g, 25.9 mmol) wasadded thionyl chloride (100 mL) the mixture was refluxed for 2 hr. andevaporated in vacuo to give the product (5.15 g, 100%). 1H-NMR (400 MHz,CDCl3): δ 9.47 (s, 1H), 8.16 (d, J=8.4 Hz, 1H), 8.03 (d, J=7.2 Hz, 1H),7.81 (t, J=8.0 Hz, 1H).

8-chloro-4-(4-(3-chlorophenyl)piperazin-1-yl)cinnoline

To a solution of 4-bromo-8-chlorocinnoline (25 mg, 0.10 mmol) and1-(3-chlorophenyl)piperazine HCl (23.9 g, 0.10 mmol) indimethylformamide (1 mL) was added potassium carbonate (42.6 mg, 0.31mmol). The reaction was stirred at 60° C. overnight. Water was added tothe mixture to precipitate the product, which was collected byfiltration or centrifugation. The product was washed with methanol anddried in vacuo to give the product (28 mg, 75%). Mass calculated forC₁₈H₁₆Cl₂N₄ 358.0751; Mass observed by HR-MS (ESI+) 359.0825 (M+H).1H-NMR (400 MHz; CDCl3): δ 9.07 (s, 1H), 7.93 (dd, J=8.5, 1.0 Hz, 1H),7.89 (dd, J=7.4, 1.0 Hz, 1H), 7.60 (dd, J=8.4, 7.5 Hz, 1H), 7.23 (d,J=8.1 Hz, 1H), 6.97 (t, J=2.1 Hz, 1H), 6.92-6.86 (m, 2H), 3.54 (dd,J=6.8, 3.1 Hz, 4H), 3.49 (dd, J=6.8, 2.9 Hz, 4H).

2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol

A solution of 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol (12.5 g, 74.1 mmol)and sodium azide (7.25 g, 111.5 mmol) in DMF (125 mL) was allowed tostir at 100° C. overnight. The mixture was filtered and evaporated togive 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol. A solution of the crudematerial and 10% Pd/C (1.12 g) in methanol (100 mL) was hydrogenated (1atm) overnight. The mixture was filtered, evaporated and purified viaflash chromatography (0-20% MeOH/DCM) to give the amine (3.53 g, 32%over 2 steps) 1H-NMR (400 MHz; CDCl3): δ 3.75-3.71 (dd, 2H), 3.69-3.64(m, 2H), 3.64-3.61 (m, 2H), 3.59-3.53 (m, 2H), 3.48-3.45 (m, 2H), 2.88(t, J=5.1 Hz, 2H).

benzyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate

To a solution of 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol (3.53 g, 23.7mmol) and diisopropylethylamine (8.2 mL, 47.3 mmol) in dichloromethane(75 mL) at 0° C. was added benzyl chloroformate (5 mL, 35.5). Thesolution was allowed warm to room temperature and stirred for 2 hours.The mixture was evaporated and purified by flash chromatography (20-100%EtOAc/hexanes) to give the benzyl carbamate (3 g, 45%). 1H-NMR (400 MHz;CDCl3): δ 7.37-7.31 (m, 5H), 5.37 (s, 1H), 5.14-5.09 (s, 2H), 3.72-3.71(m, 2H), 3.64-3.59 (m, 8H), 3.40 (q, J=5.2 Hz, 2H).

3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl-4-methylbenzenesulfonate

To a solution of benzyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate (3g, 10.6 mmol) and triethylamine (3 mL, 2 eq.) in dichloromethane (50 mL)was added tosyl chloride (2.4 g, 12.7 mmol). The solution was allowed tostir overnight, was evaporated and purified by flash chromatography(20-100% EtOAc/hexanes) to give the tosylate (2.85 g, 62%). 1H-NMR (400MHz; CDCl3): δ 7.80 (s, 1H), 7.78 (s, 1H), 7.36 (m, 5H), 7.33 (s, 1H),7.31 (s, 1H), 5.20 (s, 1H), 5.09 (s, 2H), 4.15 (t, J=4.7 Hz, 2H), 3.67(t, J=4.8 Hz, 2H), 3.56 (d, J=2.3 Hz, 2H), 3.52 (t, J=5.0 Hz, 2H), 3.37(q, J=5.3 Hz, 2H), 2.43 (s, 3H).

benzyl (2-(2-(2-(3-bromo-5-chlorophenoxy)ethoxy)ethoxy)ethyl)carbamate

To a solution of 3-bromo-5-chlorophenol (2.85 g, 6.5 mmol) in DMF (30mL) at 0° C. was added sodium hydride (348 mg, 13 mmol). After stirringfor 10 minutes 3-oxo-1-phenyl-2,7, 10-trioxa-4-azadodecan-12-yl4-methylbenzenesulfonate (3.01 g, 6.5 mmol) was added. The mixture wasallowed to stir overnight at room temperature. The mixture wasevaporated in vacuo and purified by flash chromatography to give theproduct (1.67 g, 84% brsm) 1H-NMR (400 MHz; CDCl3): δ 7.35-7.30 (m, 4H),7.10 (s, 1H), 6.96 (s, 1H), 6.84 (s, 1H), 5.25 (s, 1H), 5.09 (s, 2H),4.07 (t, J=4.4 Hz, 2H), 3.81 (t, J=4.7 Hz, 2H), 3.69-3.66 (m, 2H),3.64-3.62 (m, 2H), 3.57 (t, J=5.1 Hz, 2H), 3.40 (q, J=5.0 Hz, 2H).

tert-butyl4-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-5-chlorophenyl)piperazine-1-carboxylate

A solution of benzyl(2-(2-(2-(3-bromo-5-chlorophenoxy)ethoxy)ethoxy)ethyl)carbamate (1.67 g,3.5 mmol), tert-butyl piperazine-1-carboxylate (658 mg, 1 eq.),Tris(dibenzylideneacetone)dipalladium (97 mg, 3 mol %),Bis(diphenylphosphino)-1,1′-binaphthalene (198 mg, 9 mol %), sodiumtert-butoxide (679 mg, 7.1 mmol) in toluene (30 mL) was allowed to stirat 90° C. overnight. The mixture was purified by flash chromatography(400/o EtOAc/hexanes then 10% MeOH/DCM) to give the product (1.31 g,84%). 1H-NMR (400 MHz; CDCl3): δ 6.48 (s, 1H), 6.40 (s, 1H), 6.34 (s,1H), 4.08 (t, J=4.7 Hz, 2H), 3.81 (t, J=4.8 Hz, 2H), 3.70-3.68 (m, 2H),3.65-3.62 (m, 2H), 3.56 (t, J=5.1 Hz, 2H), 3.52 (t, J=5.2 Hz, 2H), 3.42(t, J=5.1 Hz, 2H), 3.10 (t, J=5.0 Hz, 2H), 2.92 (t, J=5.3 Hz, 2H), 2.83(t, J=5.0 Hz, 2H), 2.14 (s, 2H), 1.44 (s, 9H).

benzyl tert-butyl4-(3-chloro-5-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)phenyl)piperazine-1-carboxylate

To a solution of tert-butyl4-(3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-5-chlorophenyl)piperazine-1-carboxylate(1.31 g crude, 3 mmol) and diisopropylethylamine (1 mL, 5.9 mmol) indichloromethane (29 mL) at 0° C. was added benzyl chloroformate (0.62mL, 4.4 mmol). The solution was allowed warm to room temperature andstirred for 2 hours. The mixture was evaporated and purified by flashchromatography to give the benzyl carbamate (1.0 g, 59%). 1H-NMR (400MHz; CDCl3): δ 7.37-7.32 (m, 5H), 6.50 (s, 1H), 6.40 (s, 1H), 6.33 (s,1H), 5.26 (s, 1H), 5.09 (s, 2H), 4.07 (t, J=4.3 Hz, 2H), 3.81 (t, 1=4.8Hz, 2H), 3.70-3.67 (m, 2H), 3.65-3.62 (m, 2H), 3.59-3.51 (m, 4H), 3.39(q, J=5.5 Hz, 2H), 3.11 (t, J=5.0 Hz, 3H), 1.48 (s, 9H).

benzyl(2-(2-(2-(3-chloro-5-(piperazin-1-yl)phenoxy)ethoxy)ethoxy)ethyl)carbamate

To a solution of tert-butyl4-(3-chloro-5-((3-oxo-1-phenyl-2,7,10-trioxa-4-azadodecan-12-yl)oxy)phenyl)piperazine-1-carboxylate(1 g, 1.7 mmol) at 0° C. in dichloromethane (25 mL) was addedtrifluoroacetic acid (5 mL). The mixture was stirred for an hour and wasevaporated to give the amine (1.39 g) as the TFA salt and was taken oncrude.

benzyl(2-(2-(2-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)ethoxy)ethoxy)ethyl)carbamate

To a solution of crude(2-(2-(2-(3-chloro-5-(piperazin-1-yl)phenoxy)ethoxy)ethoxy)ethyl)carbamate(1.08 g, 2.3 mmol) in DMF (20 mL) was added potassium carbonate (1.26 g,6.8 mmol) and 4-bromo-8-chlorocinnoline (666 mg, 2.7 mmol). The mixturewas allowed to stir at 60° C. overnight and was then filtered andevaporated in vacuo at 60° C. The crude material was purified via flashchromatography (50-100% EtOAc/Hexanes) to give the product (561 mg, 38%)as a white solid. 1H-NMR (400 MHz; CDCl3): δ 9.02 (s, 1H), 7.88 (dd,J=12.1, 8.0 Hz, 2H), 7.57 (t, J=7.9 Hz, 1H), 7.32-7.27 (m, 5H), 6.57 (s,1H), 6.45 (s, 1H), 6.41 (s, 1H), 5.27 (s, 1H), 5.07 (s, 2H), 4.08 (t,J=4.8 Hz, 2H), 3.81 (t, J=4.7 Hz, 2H), 3.68 (dd, J=6.0, 3.1 Hz, 2H),3.63 (dd, J=5.7, 3.1 Hz, 2H), 3.56 (t, J=5.0 Hz, 2H), 3.50-3.46 (m, 4H),3.44-3.41 (m, 4H), 3.39-3.36 (m, 2H).

2-(2-(2-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)ethoxy)ethoxy)ethan-1-amine

To a solution of benzyl(2-(2-(2-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)ethoxy)ethoxy)ethyl)carbamate(80 mg, 0.12 mmol) in MeOH (10 mL) was added 10% Pd/C (40 mg). Themixture was allowed to stir under hydrogen at 1 atm until complete byTLC (˜30 min). The mixture was filtered an then evaporated and purifiedby HPLC to give the product (58 mg, 92%). Mass calculated forC₂₄H₂₉Cl₂N₅O₃ 505.1647; Mass observed by HR-MS (ESI+) 506.1721 (M+H).1H-NMR (400 MHz; CD3OD): δ 9.03 (s, 1H), 8.08 (d, J=8.5 Hz, 1H), 7.96(d, J=7.5 Hz, 1H), 7.68 (t, J=8.0 Hz, 1H), 6.64 (s, 1H), 6.51 (s, 1H),6.47 (s, 1H), 4.11 (t, =4.6 Hz, 2H), 3.82 (t, J=4.5 Hz, 2H), 3.70 (dd,J=5.6, 3.3 Hz, 2H), 3.65-3.63 (m, 2H), 3.62-3.60 (m, 4H), 3.48-3.46 (m,4H), 2.80 (t, J=5.3 Hz, 2H).

N-((S)-1-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)-11-(2-(3-methyl-3H-diazirin-3-yl)ethyl)-10,13-dioxo-3,6,16,19-tetraoxa-9,12-diazahenicosan-21-yl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide(1-PAP)

To a solution of biotin-PEG2-NHS (19.8 mg, 0.043 mmol) in DMF (2 mL) wasadded (S)-2-amino-4-(3-methyl-3H-diazirin-3-yl)butanoic acid (1(photo-methionine), 6.2 mg, 0.039). The mixture was allowed to stir for1 hour in a dark foil wrapped vial (or until complete by HPLC) and2-(2-(2-(3-chloro-5-(4-(8-chlorocinnolin-4-yl)piperazin-1-yl)phenoxy)ethoxy)ethoxy)ethan-1-amine(2, 20 mg, 0.039 mmol) and HATU (22.5 mg, 0.059 mmol) were added. Themixture was allowed to stir overnight and was purified by HPLC to givethe product (5 mg, 12%). Mass calculated for C₄₇H₆₅Cl₂N₁₁O₉S 1029.4064,Mass observed by HR-MS (ESI+) 1030.4139 (M+H). 1H-NMR (400 MHz;DMSO-d6): δ 9.11 (s, 1H), 8.04 (d, J=8.8 Hz, 1H), 8.02 (d, J=7.4 Hz,1H), 7.94 (d, J=8.3 Hz, 1H), 7.90 (t, J=5.8 Hz, 1H), 7.79 (t, J=5.7 Hz,1H), 7.70 (t, J=8.1 Hz, 1H), 6.63 (s, 1H), 6.49 (s, 1H), 6.45 (s, 1H),6.38 (s, 1H), 6.32 (s, 1H), 4.28 (t, J=3.6 Hz, 1H), 4.19-4.14 (m, 1H),4.11-4.06 (m, 3H), 3.70 (t, J=4.3 Hz, 2H), 3.61-3.11 (m, 27H), 3.08-3.02(m, 1H), 2.79 (dd, J=12.6, 5.0 Hz, 1H), 2.03 (t, J=7.2 Hz, 2H),1.50-1.40 (m, 2H), 1.25-1.21 (m, 6H), 0.93 (s, 3H), 0.83 (t, J=6.1 Hz,2H).

Example 6: Binding to Vimentin

To identify the relevant cellular target of Compound 1, we incubatedlive FOXC2-HMLER cells with 2.5 μM 1-PAP, as prepared in Example 5, for15 minutes and subjected these cells to UV irradiation. A samplecontaining both 1-PAP and a 20-fold molar excess of free Compound 1 wasused to distinguish specific labeling events. Surprisingly, ammoniumsulfate fractionation of the labeled lysate followed by Western blottingfor biotin revealed an abundant band in the 20% fraction whose intensitywas dramatically increased in the presence of Compound 1 competition.

We then showed that 1-PAP labels recombinant full length VIM in vitro.VIM consists of a long rod domain, which forms a coiled coil in thepresence of other VIM molecules, as well as head and tail domains. Toidentify which portion of the protein was the relevant site of 1-PAPlabeling, we expressed these domains as GST fusion proteins. In vitrolabeling experiments indicated that 1-PAP binds specifically andpotently to the rod domain of VIM. VIM is one of four type IIIintermediate filament proteins, whose members additionally includePeripherin, glial fibrillary acidic protein (GFAP), and Desmin, whichshare 59%, 61%, and 62% sequence similarity to VIM respectively (SIMalignment, ExPASy) (E. Fuchs et al., Annu. Rev. Biochem. 63 (1994)345-382). In vitro labeling experiments with recombinant preparations ofthese proteins revealed 1-PAP labeled VIM exclusively, indicating thespecificity of this interaction relative to closely related filamentousproteins.

Withaferin A (WIF-A), a steroidal lactone natural product derived fromWithania somnifera, was previously reported as a potent inhibitor ofcancer growth (G. Lahat et al., PLoS One 5 (2010) e10105; B. Grin, PLoSOne 7 (2012) e39065). Early mechanism of action studies suggested thatWIF-A inhibits the activities of VIM through covalent modification ofthe single cysteine residue present on the rod domain of the protein (P.Bargagna-Mohan, et al., Chem. Biol. 14 (2007) 623-634). WIF-A treatmentwas found to be selectively cytotoxic to FOXC2-HMLER cells with an˜10-fold cytotoxic index relative to control cells, confirming thenotion that targeting VIM is lethal to mesenchymally transformed cells.We reasoned that toxicity to both cell lines at doses above 200 nM mightbe explained by WIF-A's reported off target inhibitory activities whichinclude the covalent modification of GFAP, β-Tubulin, NF-κB, and Sp1 (P.Bargagna-Mohan et al., J. Biol. Chem. 285 (2010) 7657-7669; M. L. Antonyet al., J. Biol. Chem. 289 (2014) 1852-1865; K. Heyninck et al.,Biochem. Pharmacol. 91 (2014) 501-509). Although these off targetinteractions indicate WIF-A is unsuitable as a selective chemical probeof VIM, we nevertheless used it as a positive control in certain assaysfor which WIF-A has been previously reported to inhibit VIM function.

WIF-A is thought to induce cell death of VIM-expressing cancer cells, atleast in part, by inducing filamentous network collapse and degradationof VIM (B. Grin (2012) and G. Lahat (2010, supra). To monitor theassembly status of the VIM filamentous architecture, we performedimmunofluorescent analyses of human umbilical endothelial cells (HUVECs)that were treated with Compound 1 and WIF-A, which have been shown todisplay a high elaborated VIM architecture sensitive to WIF-A treatment(P. Bargagna (2007), supra).

These studies revealed that one-hour treatment with Compound 1 and WIF-Apromoted the destruction of fine VIM-containing filamentous structuresand the contraction of the VIM apparatus relative to the boundary of thecell. Additionally, WIF-A and Compound 1 induced rapid morphologicalchanges in the appearance of FOXC2-HMLER cells as determined byimmunofluorescent analysis for VIM staining, a result consistent withthe idea that the previously described morphological changes are due toVIM reorganization.

Further, treatment of FOXC2-HMLER cells with Compound 1 dramaticallyreduced the appearance of dibromobimane-crosslinked dimeric VIM proteincontent, as shown by Western blotting, by procedures previously used tomonitor the filamentous status of VIM in cells and in solution (D.Perez-Sala, et al. Nat. Commun. 6 (2015) 7287. Additionally, treatmentof FOXC2-HMLER cells with Compound 1 or WIF-A induced the dose-dependentaccumulation of lower molecular weight VIM degradation products asvisualized by Western blotting.

WIF-A has also been reported to induce the degradation of VIM protein bya ubiquitin proteasome mediated mechanism (P. Bargagna-Mohan (2007),supra). Analyzing the protein content of FLAG-immunoprecipitated VIMfrom HEK293T cells, we observed a clear accumulation of high molecularweight ubiquitinated VIM species. This result is consistent with theidea that both compounds trigger the degradation of VIM through aubiquitin mediated mechanism.

Example 7: Modulation of Phosphorylation State of Vimentin

We performed Western blotting analysis of FOXC2-HMLER cells treated for24 hours with Compound 1 with commercially available antibodiestargeting specific phosphorylated VIM species (P-S39, P-S56, P-S83).Treatment by Compound 1 induced a modest increase in P-S39- andP-S83-VIM protein content but led to a marked increase in the steadystate levels of P-S56-VIM (FIG. 4).

Immunofluorescent staining experiments for P-S56-VIM corroborated thisobservation, with Compound 1 treatment resulting in a concentrationdependent increase in P-S56-VIM positive FOXC2-HMLER cells (FIG. 5). Incontrast, only a small fraction of cells undergoing mitosis stainedpositive for P-S56-VIM in DMSO-treated controls, consistent withprevious reports identifying phosphorylation at S56 as a modificationcatalyzed by cyclin dependent kinase 1 (CDK1; FIG. 6) (T. Yamaguchi, T.et al., J. Cell Biol. 171 (2005) 431-436.

To determine if this increase in P-S56-VIM protein content might beresponsible for FiVel's ability to induce multinucleation, we performedtransient overexpression experiments with vectors encoding FLAG-taggedwild type VIM (VIM-wt-FLAG) or a phospho-mimetic mutant of VIM at S56(VIM-S56E-FLAG) in FOXC2-HMLER and HEK293T cells. 72-hour expression ofthe VIM-S56E-FLAG transgene was found to induce multinucleation in bothcell lines. We additionally overexpressed these transgenes by stablelentiviral delivery in FOXC2-HMLER cells. Whereas VIM-wt-FLAG expressingcells grew similarly to dTomato-expressing control cells over 7 days,VIM-S56E-FLAG cells were found to be replication incompetent over thisperiod. Together, these results suggest that a specific, sustainedphosphorylation modification on VIM is sufficient to recapitulate theactivity of Compound 1.

Because Compound 1 induces a hyper-phosphorylated VIM phenotype, wesought to determine if Compound 1's engagement of VIM might interfere atan alternative stage of mitosis. We therefore performed time courseconfocal imaging studies of Compound 1 treated FOXC2-HMLER cells aftertheir release from thymidine blocking-based cell cycle synchronization.While Compound 1 treated cells were found to condense their chromosomesduring anaphase normally, Compound 1 treated cells exhibited a number ofaltered phenotypes during metaphase.

Compound 1 treatment resulted in a collapsed VIM filamentous structure,which appeared more closely associated to mitotic spindle poles whencompared to DMSO treated controls (FIG. 7). Additionally, we observedthat Compound 1 treatment inhibited the ability of chromosomes to alignto the metaphase plate, frequently occupying regions distal to thespindle pole.

Compound 1 treatment also disrupted the ability of β-tubulin tofaithfully form the fine spindle microtubules of the mitotic spindle,instead resulting in a compressed phenotype with radiating projectionsconnecting unaligned chromosomes. To assess the uniqueness of thisphenotype, we evaluated the metaphase phenotypes of other chemicalinhibitors which have shown to alter mitotic progression. These includedinhibitors of centromere-associated protein E (CENP-E, also calledkinesin-7, GSK92395), kinesin spindle protein (KSP, also called Eg5,Ispinesib), polo like kinases (PLK1/2/3, BI2536), and aurora kinases(AURKA/B/C, VX-680). Treating thymidine-synced FOXC2-HMLER cells withthese inhibitors revealed that the majority of these compounds did notinduce phenotypes similar to Compound 1, with the exception of GSK92395,which also demonstrated an unaligned chromosome phenotype in line withCENP-E's reported function of aligning chromosomes to the metaphaseplate (K. W. Wood et al., Proc. Natl. Acad. Sci. USA 107 (2010)5839-5844). However, GSK92395 treatment did not induce VIM filamentouscollapse or alter the morphology of spindle tubulin. Taken together,these results suggest that Compound 1 induces mitotic failure andeventual multinucleation through a novel mechanism which involvesinterfering with the metaphase organization of chromosomes and thespindle apparatus.

WIF-A induced a significant increase in multinucleated FOXC2-HMLER cellsat concentrations (250-500 nM) below those at which it induced anapoptotic, non-adherent phenotype during this treatment period (24hours). Additionally, treating thymidine synched FOXC2-HMLER cells withWIF-A resulted in the characteristic appearance of unaligned chromosomesduring metaphase, although not all metaphase plates displayed anunaligned phenotype as observed with a maximally efficacious dose ofCompound 1. While differences in target engagement or off-targetmodification might explain this difference, together these dataidentifies the chemical targeting of VIM as a generalizable path forinterfering with mitotic progression.

1. A method for treating a patient suffering from amesenchymally-derived or mesenchymally-transformed cancer, comprisingadministering to the subject a therapeutically effective amount of acompound, or a pharmaceutically acceptable salt thereof, according toany one of Formulae I-IV:

wherein R^(1A) in each instance is independently selected from the groupconsisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, and C₁-C₆-alkoxy, halo, CN,—S—C₁-C₆-alkyl, —C(O)N(R^(2A))₂; R^(2A) in each instance isindependently selected from H and C₁-C₆-alkyl; each ---, if present,represents a single bond; n is 0, 1, 2, or 3; Het^(A) is 6-memberedmonocyclic or 9- to 10-membered bicyclic heteroaryl, wherein 1 to 3 ringmembers are N; Het^(A) and any alkyl, alkenyl, alkoxy, and aryl isoptionally substituted with 1-3 substituents selected from the groupconsisting of C₁-C₆-alkyl, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and—C(O)O—C₁-C₆-alkyl;

wherein R^(1B) and R^(2B) are independently selected from the groupconsisting of H, C₁-C₆-alkyl, and C₂-C₆-alkenyl; R^(3B) and R^(4B) areindependently selected from the group consisting of H, C₁-C₆-alkyl,C₂-C₆-alkenyl, and C₁-C₆-alkoxy; or R^(3A) and R^(4A), together with thecarbon atoms to which they are attached, represent a fused 5- to6-membered heterocycle, wherein 1 to 2 ring members are selected fromNR^(1B), O, and S; Ar^(1B) and Ar^(2B) are independently C₆-C₁₀-aryl;any alkyl, alkenyl, alkoxy, aryl, and heteroaryl is optionallysubstituted with one or more substituents selected from the groupconsisting of C₁-C₆-alkyl, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and—C(O)O—C₁-C₆-alkyl;

wherein R^(1C), R^(2C), and R^(3C) are independently selected from thegroup consisting of H, C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, CN,C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and —C(O)O—C₁-C₆-alkyl; f is 0, 1, 2, or3; o, p, and q are independently selected from 0, 1, 2, 3, 4, and 5; Xand Y are independently selected from the group consisting of a bond,—NH—, and —CH₂—; Cy^(1C) is selected from the group consisting ofC₃-C₈-cycloalkyl and 3- to 7-membered heterocyclo wherein 1-3 ringmembers are selected from N, O, and S; any alkyl, alkenyl, alkoxy, aryl,and heterocyclo is optionally substituted with 1-3 substituents selectedfrom the group consisting of C₁-C₆-alkyl, halo, CN, C₁-C₆-alkoxy,—C(O)C₁-C₆-alkyl, and —C(O)O—C₁-C₆-alkyl;

wherein Cy^(1D) is a 5- to 6-membered heteroaryl (wherein 1-3 ringmembers are N) or C₆-C₁₀-aryl, wherein Cy^(1D) is optionally substitutedby 1-3 R^(1D); Cy^(2D) is a 5- to 6-membered heteroaryl (wherein 1-3ring members are N) or C₆-C₁₀-aryl, wherein Cy^(2D) is optionallysubstituted by 1-3 R^(2D); R^(1D) in each instance is independentlyselected from the group consisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, halo,C₆-C₁₀-aryl; R^(2D) in each instance is independently is selected fromthe group consisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, C₆-C₁₀-aryl;R^(1D) and R^(2D) are not simultaneously C₆-C₁₀-aryl: R^(3D) in eachinstance is independently selected from the group consisting ofC₁-C₆-alkyl, C₂-C₆-alkenyl, and halo; r is 0, 1, 2, or 3; and any alkyl,alkenyl, alkoxy, heteroaryl, and aryl is optionally substituted with oneor more substituents selected from the group consisting of C₁-C₆-alkyl,halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and —C(O)O—C₁-C₆-alkyl.
 2. Themethod according to claim 1, wherein the cancer is a sarcoma or breastcancer.
 3. The method according to claim 1, wherein the cancer is breastcancer.
 4. The method according to claim 1, wherein the cancer is asarcoma.
 5. The method according to claim 1, wherein the compound or apharmaceutically acceptable salt thereof is a compound according toFormula I.
 6. The method according to claim 5, wherein each --- isabsent; and Het^(A) is a 9- to 10-membered bicyclic heteroaryl.
 7. Themethod according to claim 6, wherein Het^(A) is substituted by 1-3 halo.8. The method according to claim 1, wherein the compound or apharmaceutically acceptable salt thereof is a compound according toFormula II.
 9. The method according to claim 8, wherein Ar^(1B) andAr^(2B) are independently optionally substituted phenyl.
 10. The methodaccording to claim 9, wherein each of R^(1B) and R^(2B) is H.
 11. Themethod according to claim 1, wherein the compound or a pharmaceuticallyacceptable salt thereof is a compound according to Formula III.
 12. Themethod according to claim 11, wherein X and Y each is a bond; f is 0;and Cy^(1C) is an optionally substituted 3- to 7-membered heterocyclowherein 1-3 ring members are N.
 13. The method according to claim 12,wherein Cy^(1C) is optionally substituted piperazinyl.
 14. The methodaccording to claim 1, wherein the compound or a pharmaceuticallyacceptable salt thereof is a compound according to Formula IV.
 15. Themethod according to claim 14, wherein Cy^(1D) is an optionallysubstituted 5- to 6-membered heteroaryl (wherein 1-3 ring members areN); and Cy^(2D) is an optionally substituted 5- to 6-membered heteroaryl(wherein 1-3 ring members are N).
 16. The method according to claim 15,wherein Cy^(1D) is substituted by one R^(1D) that is optionallysubstituted C₆-C₁₀-aryl.
 17. The method according to claim 15, whereinCy^(2D) is substituted by one R^(2D) that is optionally substitutedC₆-C₁₀-aryl.
 18. The method according to claim 1, wherein the compoundor a pharmaceutically acceptable salt thereof is one selected from thefollowing table: 1

1A

1B

1C

1D

1E

1F

1G

1SRH

1SRI

1SRJ

1SRK

1SRL

1SRM

1SRN

1SRO

1SRP

1SRQ

1SRU

1SRT

1SRU

1SRV

1SRW

1SRX

1SRY

1SRZ

1SRAA

1SRAB

1SRAC

1SRAD

1SRAE

1SRAF

1SRAG

1SRAH

1SRAI

1SRAJ

1SRAK

1SRAL

1SRAM

1SRAN


19. The method according to claim 1, wherein the compound or apharmaceutically acceptable salt thereof is one selected from thefollowing table: 2A

2

2B

2C

2D

2E

2F

2G

2H

2I

2J

2K

2L

2M

2N

2O

2P

2Q

2R

2S

2T

2U

2V

2W

2X

2Y

2Z

2AA

2AB

2AC

2AD

2AE

2AF

2AG

2AH

2AI

2AJ

2AK

2AL

2AM

2AN

2AO

2AP

2AQ

2AR

2AS

2AT

2AU

2AV

2AW

2AX

2AY

2AZ

2BA

2BB

2BC

2BD

2BE

2BF

2BG

2BH

2BI

2BJ

2BK

2BL

2BM

2BN

2BO

2BP

2BQ

2BR

2BS

2BT

2BU

2BV

2BW

2BX

2BY

2BZ

2CA

2CB

2CC

2CD

2CE

2CF

2CG

2CH

2CI

2CJ

2CK

2CL

2CM

2CN

2CO

2CP

2CQ

2CR

2CS

2CT

2CU

2CV

2CW

2CX

2CY

2CZ

2DA

2DB

2DC

2DD

2DE

2DF

2DG

2DH

2DI

2DJ

2DK

2DL

2DM

2DN

2DO

2DP

2DQ

2DR

2DS

2DT

2DU

2DV

2DW

2DX

2DY

2DZ

2EA

2EB

2EC

2ED

2EE

2EF

2EG

2EH

2EI

2EJ

2EK

2EL

2EM

2EN

2EO

2EP

2EQ

2ER

2ES

2ET

2EU

2EV

2EW

2EX

2EY

2EZ

2FA

2FB

2FC

2FD

2FE

2FF

2FG

2FH

2FI

2FJ

2FK

2FL

2FM

2FN

2FO

2FP

2FQ

2FR

2FS

2FT

2FU

2FV

2FW

2FX

2FY

2FZ

2GA

2GB

2GC

2GD

2GE

2GF

2GG

2GH

2GI

2GJ

2GK

2GL

2GM

2GN

2GO

2GP

2GQ

2GR

2GS

2GT

2GU

2GV

2GW

2GX

2GY

2GZ

2HA

2HB

2HC

2HD

2HE

2HF

2HG

2HH

2HI

2HJ

2HK

2HL

2HM

2HN

2HO

2HP

2HQ

2HR

2HS

2HT

2HU

2HV

2HW

2HX

2HY

2HZ

2IA

2IB

2IC

2ID

2IE

2IF

2IG

2IH

2II

2IJ

2IK

2IL

2IM


20. The method according to claim 1, wherein the compound or apharmaceutically acceptable salt thereof is one selected from thefollowing table: 3A

3B

3C

3D

3E

3F

3

3G

3H

3I

3J

3K

3L

3M

3N

3O

3P

3Q

3R

3S

3T

3U

3V

3W

3X

3Y

3Z

3AA

3AB

3AC

3AD

3AE

3AF

3AG

3AG

3AH

3AI

3AJ

3AK

3AL

3AM

3AN

3AO

3AP

3AQ

3AR

3AS

3AT

3AU

3AV

3AW

3AX

3AY

3AZ

3BA

3BB

3BC

3BD

3BE

3BF

3BG

3BH

3BI

3BJ

3BK

3BL

3BM

3BN

3BS

3BT

3BU

3BV

3BO

3BP

3BQ

3BR

3BW

3BX

3BY

3BZ

3CA

3CB

3CC

3CD

3CE

3CF

3CG

3CH

3CI

3CJ

3CK


21. The method according to claim 1, wherein the compound or apharmaceutically acceptable salt thereof is one selected from thefollowing table: 4A

4B

4C

4D

4E

4

4F

4G

4H

4I

4J

4K

4L

4M

4N

4O

4P

4Q

4R

4S

4T

4U

4V

4W

4X

4Y

4Z

4AA

4AB

4AC

4AD

4AE

4AF

4AG

4AH

4AI

4AJ

4AK

4AL

4AM

4AN

4AO

4AP

4AQ

4AR

4AS

4AT

4AU


22. A method of treating a subject suffering from a disease or conditionthat is characterized by the expression of vimentin, comprisingadministering to the subject a therapeutically effective amount of acompound, or a pharmaceutically acceptable salt thereof, according toany one of Formulae I-IV:

wherein R^(1A) in each instance is independently selected from the groupconsisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, and C₁-C₆-alkoxy, halo, CN,—S—C₁-C₆-alkyl, —C(O)N(R^(2A))₂; R^(2A) in each instance isindependently selected from H and C₁-C₆-alkyl; each ---, if present,represents a single bond; n is 0, 1, 2, or 3; Het^(A) is 6-memberedmonocyclic or 9- to 10-membered bicyclic heteroaryl, wherein 1 to 3 ringmembers are N; Het^(A) and any alkyl, alkenyl, alkoxy, and aryl isoptionally substituted with 1-3 substituents selected from the groupconsisting of C₁-C₆-alkyl, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and—C(O)O—C₁-C₆-alkyl;

wherein R^(1B) and R^(2B) are independently selected from the groupconsisting of H, C₁-C₆-alkyl, and C₂-C₆-alkenyl; R^(3B) and R^(4B) areindependently selected from the group consisting of H, C₁-C₆-alkyl,C₂-C₆-alkenyl, and C₁-C₆-alkoxy; or R^(3A) and R^(4A), together with thecarbon atoms to which they are attached, represent a fused 5- to6-membered heterocycle, wherein 1 to 2 ring members are selected fromNR^(1B), O, and S; Ar^(1B) and Ar² are independently C₆-C₁₀-aryl; anyalkyl, alkenyl, alkoxy, aryl, and heteroaryl is optionally substitutedwith one or more substituents selected from the group consisting ofC₁-C₆-alkyl, halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and—C(O)O—C₁-C₆-alkyl;

wherein R^(1C), R^(2C), and R^(3C) are independently selected from thegroup consisting of H, C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, CN,C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and —C(O)O—C₁-C₆-alkyl; f is 0, 1, 2, or3; o, p, and q are independently selected from 0, 1, 2, 3, 4, and 5; Xand Y are independently selected from the group consisting of a bond,—NH—, and —CH₂—; Cy^(1C) is selected from the group consisting ofC₃-C₈-cycloalkyl and 3- to 7-membered hetercyclo wherein 1-3 ringmembers are selected from N, O, and S; any alkyl, alkenyl, alkoxy, aryl,and heterocyclo is optionally substituted with 1-3 substituents selectedfrom the group consisting of C₁-C₆-alkyl, halo, CN, C₁-C₆-alkoxy,—C(O)C₁-C₆-alkyl, and —C(O)O—C₁-C₆-alkyl;

wherein Cy^(1D) is a 5- to 6-membered heteroaryl (wherein 1-3 ringmembers are N) or C₆-C₁₀-aryl, wherein Cy^(1D) is optionally substitutedby 1-3 R^(1D); Cy^(2D) is a 5- to 6-membered heteroaryl (wherein 1-3ring members are N) or C₆-C₁₀-aryl, wherein Cy^(2D) is optionallysubstituted by 1-3 R^(2D); R^(1D) in each instance is independentlyselected from the group consisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, halo,C₆-C₁₀-aryl; R^(2D) in each instance is independently is selected fromthe group consisting of C₁-C₆-alkyl, C₂-C₆-alkenyl, halo, C₆-C₁₀-aryl;R^(1D) and R^(2D) are not simultaneously C₆-C₁₀-aryl; R^(3D) in eachinstance is independently selected from the group consisting ofC₁-C₆-alkyl, C₂-C₆-alkenyl, and halo; r is 0, 1, 2, or 3; and any alkyl,alkenyl, alkoxy, heteroaryl, and aryl is optionally substituted with oneor more substituents selected from the group consisting of C₁-C₆-alkyl,halo, CN, C₁-C₆-alkoxy, —C(O)C₁-C₆-alkyl, and —C(O)O—C₁-C₆-alkyl. 23.The method according to claim 22, wherein the disease or condition isselected from mesenchymally-derived or mesenchymally-transformedcancers, autoimmune diseases, neuropathies, inflammatory diseases, andcataracts.
 24. The method according to claim 22, wherein the disease orcondition is a mesenchymally-derived or mesenchymally-transformedcancer.
 25. The method according to claim 22, wherein the disease orcondition is a neuropathy.
 26. The method according to claim 25, whereinthe neuropathy is giant axonal neuropathy (GAN).
 27. (canceled)